Eukaryotic ion channels play crucial roles in many aspects of cell physiology. Understanding the structure and function of these proteins relies upon the availability of specific, high-affinity probes. The long-term goals of this laboratory are to determine the mechanisms of permeation and gating in two structurally unrelated, clinically relevant anion channels: the CFTR channel and the CIC family of channels. CFTR is the locus of the primary defect in cystic fibrosis (CF), the most common lethal, autosomal recessive disease among Caucasians, affecting approximately 30,000 Americans; CFTR is also crucial to the pathophysiologies of secretory diarrhea and polycystic kidney disease (PKD). C1C channels are nearly ubiquitous, and play important roles in several segments of the nephron. Despite significant study, including the recent demonstration of the crystal structure of two prokaryotic C1C proteins, the structures involved in permeation (for CFTR and C1Cs) and gating (for CICs) are not clear, partly due to lack of appropriate tools. Peptide toxins have proven to be among the most selective and useful tools for the study of cation-permeable channels. However, no such peptide toxins have been shown to interact with anion channels of known molecular identity. Preliminary data from this laboratory has now shown that scorpion venom contains a peptide component, or components, that inhibit(s) CFTR and C1C-2channels, albeit with different mechanism. This R21 proposal seeks to take the initial steps to develop these exciting observations, by identifying the active toxin or toxins and using it/them to study the architecture and mechanisms of gating in these ion channels. Specific Aim #1 is to gather initial information on inhibition by venom, then use this to form bioassays to guide the chromatographic separation and identification of the active component(s). Specific Aim #2 will then use the isolated toxin(s) as a high-affinity probe to study CFTR and CIC-2 channels in exquisite detail. The experimental plan combines the significant expertise of this laboratory in using structure/function approaches (by electrophysiological methods) to study CI channels with the expertise of collaborators in using state-of-the-art chromatographic and spectrometric approaches for the identification of bioactive macromolecules. The proposed exploratory work will allow the study of these important channel proteins in ways heretofore impossible, and will likely aid in the design of novel therapies for CF, secretory diarrhea, polycystic kidney disease, and other nephropathies.